Probe for detecting a highly ordered structural site of a single stranded nucleic acid of a gene, and a method and a device for detecting the same

ABSTRACT

The invention provides a probe for detecting a highly ordered structural site of a nucleic acid of a gene by specifically binding with the structural site to generate an electrochemical response. The inventive probe comprises a cyclic ligand containing ferrocenyl group and a DNA threading intercalating moiety, such as 1,4,5,8-tetrasubstituted naphthalene, 9,10-disubstituted anthracene, and 1,5-disubstituted anthraquinone. Current of the cyclic ligand is not observed due to interaction such as stacking or so called charge transfer between ferrocenyl group and the DNA threading intercalating moiety in conventional electrolyte. The binding of the ligand with a highly ordered structural site of a single stranded nucleic acid, where nucleic base inserts between the cavity of cyclic ligand, inhibits the intramolecular interaction of the ligand to convert the ligand into its electrically active form, and as a result, current is observed.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The invention relates to a probe for detecting a highly orderedstructural site of a single stranded nucleic acid of a gene, a methodfor detecting the same using the probe, and a device for detecting thesame.

[0003] 2. Related Arts

[0004] Sensors and sensing techniques have been utilized in every fieldof industries. In particular in biotechnology field, a high-sensitivitysensor system utilizing an enzyme reaction has been established.Recently, importance of gene sensing is increasing in applications suchas gene therapy and gene diagnosis. Up to now, “DNA probe method” hasbeen accepted as such gene sensing method.

[0005] A highly ordered structural site of a single stranded nucleicacid is a region located in a part of high-order structure of a DNA orRNA where the bases of the single stranded nucleic acids are notstacked, the region including a mismatch structure of an oncogenic DNA,a hairpin structure of a viral RNA and a bulge.

[0006] Such DNA probe method is mostly carried out by manual operations.Consequently, it has been demanded a gene sensor other than the DNAprobe type sensor for detecting efficiently a specialized nucleic acidregion of a gene. However, such sensor has not yet been reported orpatented.

[0007] The object of the invention is to provide a detection probe fordetecting a highly ordered structural site of a single stranded nucleicacid of a gene, and to provide a detection method and device using suchprobe.

[0008] The invention provides a probe for detecting a highly orderedstructural site of a single stranded nucleic acid of a gene byspecifically binding with the site to generate an electrochemicalresponse, the probe comprising a cyclic ligand containing ferrocenylgroup and a DNA threading intercalating moiety.

[0009] The DNA threading intercalating moiety is a moiety or residualgroup derived from a DNA threading intercalating compound. The DNAthreading intercalating compound is a compound which can intercalate orslide between adjacent base pairs of a double stranded DNA with its twosubstituents projecting out of the major and minor groovesimultaneously.

[0010] The invention also provides a method for detecting a highlyordered structural site of a single stranded nucleic acid of a gene, themethod comprising:

[0011] contacting a gene to be detected with the above probe to generatean electrochemical response; and

[0012] detecting the electrochemical response.

[0013] The inventors successfully developed a cyclic ligand, whosetarget is a highly ordered structural site of a single stranded nucleicacid of a gene, generating an electrochemical response only when suchsite is present in the gene. The probe may thereby provide a system forsensing a highly ordered structural site of a single stranded nucleicacid of a DNA or RNA with efficiency and high sensitivity. Moreover, inprior DNA probes, different DNA probes are required and providedresponding to different target genes. On the contrary, the inventivedetection probe may be applied to every gene having a highly orderedstructural site of a single stranded nucleic acid.

[0014] The site of a gene is a part of a high-order structure in a DNAor RNA, including a mismatch structure frequently seen in an oncogenicDNA, an RNA hairpin structure of a virus, and a bulge structure. Suchstructural site comprises basically a single stranded structure, whichusually coexists with a double stranded structure, causing somedeformation in the structural site compared with a common singlestranded nucleic acid structure. If the site structure is consecutive(such as in the case of a bulge or hairpin structure), it has a uniquestacking structure entirely different from that of a common singlestranded nucleic acid. For example, the onset of fragile X syndrome isdeeply influenced by repeated hairpin structures. An HIV virus alsopreserves mismatch, hairpin and bulge structures present in its TAR-RNAand REE RNA, and such structures are indispensable for expressing thefunction of a HIV virus.

[0015] In the invention, ferrocene and naphthalene diimide (naphthalenebis(dicarboximide) or naphthalene-1,8; 4,5-diimide)) are condensed toobtain a cyclic ligand. Manufacturing example of the cyclic ligand isshown in FIG. 1. This representative cyclic ligand is named “cyclicnaphthalenediimideferrocene” (referred to as “CNDIFc” below). Theinventive cyclic ligand comprises ferrocenyl group, two amide bonds orgroups bonded with both ends of ferrocenyl group, naphthalene diimidemoiety, and two connecting groups each connecting each amide bond orgroup and each N-terminal of naphthalene diimide moiety.

[0016] The DNA threading intercalating moiety may preferably comprise anaromatic group selected from a group consisting of1,4,5,8-tetrasubstituted naphthalene, 9,10-disubstituted anthracene and1,5-disubstituted anthraquinone. The connecting manner (the positions ofsubstitution) of the aromatic group plays decisive role as to whetherthe aromatic group works as the threading intercalating moiety(threading intercalator).

[0017] The cyclic ligand may further comprise two linker moiety eachhaving two terminal amino groups. Each linker moiety is connected withthe DNA threading intercalating moiety through one of its terminal aminogroups and is connected with the ferrocenyl group through the other ofits terminal amino groups.

[0018] The linker moiety may preferably be a residual group of an aminehaving two terminal amino groups. The amine may preferably compriseanother amino group and two alkyl groups. Each alkyl group is bondedwith each terminal amino group and another amino group. In this case,the another amino group may preferably be piperazinyl group, methylaminogroup, or amino group, and most preferably piperazinyl group. The alkylgroup may preferably be C1-6 alkyl group and more preferably be ethyl orpropyl group. The amine may most preferably be one of the followings.

[0019] 1,4-bis(3-aminopropyl)piperazine,1,1′-bis(3-aminopropyl)methylamine, 1,1′-bis(2-aminoethyl)amine,1,1′-bis(3-aminopropyl)amine, spermine and spermidine.

[0020] The DNA threading intercalating moiety may further comprise, inaddition to the aromatic group, carbonyl groups, iminomethylene groups(C═NR, R is hydrogen or an alkyl group), or thiocarbonyl groups (—C═S),through which the intercalating moiety may be bonded with the terminalamino groups. When the aromatic group is naphthalene, the terminal aminogroups may be bonded through carbonyl groups, iminomethylene groups, orthiocarbonyl groups to 1, 4, 5, 8 positions of naphthalene, to form, forexample, two imide groups or naphthalene-diimide moiety. When thearomatic group is anthracene, the terminal amino groups are bondedthrough carbonyl groups, iminomethylene groups, or thiocarbonyl groupsto 9 and 10 positions of anthracene. When the aromatic group isanthraquinone, the terminal amino groups are bonded through carbonylgroups, iminomethylene groups, or thiocarbonyl groups to 1 and 5positions of anthraquinone.

[0021] Ferrocenyl group and the other terminal amino group of eachlinker moiety may preferably be bonded through methylene group orcarbonyl group.

[0022] In most preferred production process of the ligand, one mole of1,4,5,8-naphthalene tetracarboxylic acid, its monoanhydride ordianhydride is reacted with two moles of a connecting compound havingtwo N-terminals (terminal amino groups) to produce one mole of a diaminobody. This diamino body has naphthalene diimide moiety and twoconnecting groups, each having one N-end (terminal amino group). Thediamino body is then reacted with ferrocene dicarboxylic acid or itsactive ester to provide a cyclic ligand.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 is a synthesis scheme of a representative example (CNDIFc)of the inventive detection probe, and

[0024]FIG. 2 is graphs showing examples of detecting a specific nucleicacid region, in which graph (a) is a cyclic voltamogram of CNDIFc usinga working electrode made of gold modified with no gene, graph (b) is acyclic voltamogram of CNDIFc using a working electrode made of goldmodified with a hairpin structure DNA, and graph (c) is a cyclicvoltamogram of CNDIFc after hybridizing the above hairpin structure withan oligonucleotide complementary with the hairpin structure DNA.

[0025] The invention will be described below in detail. The inventivedetection device has a container holding solution containing the cyclicligand, a working electrode modified with a gene, and a counterelectrode to the working electrode, wherein the working electrode andcounter electrode are dipped in the solution. The device may preferablybe provided with a reference electrode.

[0026] The working, counter and reference electrodes may preferably madeof, for example, gold, glassy carbon, or carbon. A gene to be detectedmay be immobilized onto the working electrode by means of a knownmethod. For example, when the working electrode is made of gold, a thiolgroup may be introduced in a gene to bind it onto the working electrodeby means of a gold-sulfur coordination bond. Such method for binding agene with the working electrode of gold is described, for example, by B.A. Connolly, in a publication “Nucleic acids Res.” 13, 4484, 1985.Moreover, a gene may be immobilized onto the working electrode made ofglassy carbon by oxidizing the working electrode with potassiumpermanganate to introduce a carboxylic acid on the surface of theworking electrode, and by binding the carboxylic acid with an amino acidconstituting the nucleic acid of the gene. This method is described byKelly M. Mollan and Susan R. Mikkelsen in a publication “AnalyticalChemistry” 65, 2317-2323 (1993).

[0027] Preferably, a gene to be detected as such, or after replicatingit by means of a polymerase chain reaction, is provided with a thiolgroup using mercaptoethanol and1-ethyl-3-(3′-dimethylaminopropyl)-carbodiimide (WSCI), and thenimmobilized onto an electrode of gold by chemisorption. Alternatively, agene to be detected as such, or after replicating it by means of apolymerase chain reaction using 5′-thiolated oligonucleotide as one ofprimers, is immobilized onto a gold electrode by chemisorption. Thisgold electrode functions as the working electrode. The inventiveelectrochemical determination is carried out in a cell containing theworking, counter and reference electrodes in the presence of the cyclicligand. Oxidation and reduction reactions of ferrocenyl group present inthe inventive cyclic ligand induce current, whose amplitude may providean indicator of whether a gene to be detected has a highly orderedstructural site of single stranded nucleic acid or not and/or thecontent of the site. The amplitude of the induced current may bedetected by a means or apparatus such as a cyclic voltamogram, adifferential pulse voltamogram, or a potentiostat.

[0028] Phosphate buffered saline and the other salts may be added, inaddition to the inventive cyclic ligand, to the solution. The solutionmay be adjusted as described below so that the inventive ligandspecifically binds with the site and scarcely binds with a doublestranded nucleic acid site. That is, the solution may preferably contain1 to 50 μM (micromole) of the cyclic ligand, 10 to 100 mM of phosphatebuffered saline, and 10 to 50 mM of a salt, the salt may preferablybeing a salt of an alkaline metal such as potassium or sodium.

EXAMPLES

[0029] The experimental results will be described below.

[0030] A cyclic ligand was synthesized according to the scheme of FIG.1.

[0031] (Synthesis of an Amine Body)

[0032] 2 g of 1,4,5,8-naphthalene tetracarboxylic acid dianhydride (7.45mmol) and 40 ml of 1,4-(3-aminopropyl)piperazine (190 mmol) are refluxedin tetrahydrofuran for 8 hours. After the mixture was cooled to roomtemperature, hexane was added to precipitate crystal, which was thencollected by filtration. The thus obtained crystal was dissolved in aminimum volume of chloroform and recrystallized in ether. The resultingcrystal was then removed, from which ether was vacuum-evaporated. Thethus obtained crystal was dissolved again in chloroform andrecrystallized in hexane. The resulting crystal was collected. Theamount of the crystal was 330 mg and the yield was 52 percent. Thecrystal showed the following characteristics. red-brown solid meltingpoint 300° C. ¹H-NMR chemical shifts (CDCl₃, ppm) 1.58, 1.95, 2.27-2.52,2.71, 4.28, 8.75 IR C = 0 1650 cm⁻¹

[0033] (Synthesis of an Active Ester of 1,1′-ferrocene DicarbolylicAcid)

[0034] 0.14 g (0.5 mmol) of 1,1′-ferrocene dicarboxylic acid wasdissolved in 15 ml of DMF to obtain solution, to which 0.66 g (1.5 mmol)of POB reagent and 0.21 ml (1.5 mmol) of triethylamine dissolved in 5 mlof DMF were added dropwise. The resultant solution was stirred at roomtemperature for one and a half hour to obtain reagent solutioncontaining an active ester of 1,1′-ferrocene dicarboxylic acid. Thereagent solution was used to a reaction with the above amine body.

[0035] (Production of a Cyclic Ligand)

[0036] 0.35 g (0.5 mmol) of the amine body was dissolved in 300 ml ofchloroform. To the amine body solution, the reagent solution, containingan active ester of ferrocene dicarboxylic acid, was added dropwise whilestirring the amine body solution. The resultant solution was thenstirred at room temperature for 20 hours. The reaction solution wasfiltered to obtain filtrate, which was solidified at a reduced pressureand dissolved in methanol. The methanol solution was developed by silicagel column chromatography using methanol as its developing solvent toobtain a fraction seemingly containing a cyclic ligand. Methanol wasremoved at a reduced pressure from the fraction to provide precipitate,which was then dissolved in chloroform and washed with saturated sodiumcarbonate solution. Chloroform was removed from the solution to obtaincrystal, which was dried at a reduced pressure and collected. 46 mg ofcrystal was obtained and its yield was 10 percent. The crystal showedthe following characteristics. yellow crystal melting point 300° C.¹H-NMR chemical shifts (CDCl₃, ppm) 1.56, 1.83, 1.99, 2.26, 2.45, 3.27,4.28, 4.40, 4.43, 6.85, 8.77 IR C═O 1791, 1661 cm⁻¹ elemental analysisH(%) C(%) N(%) calculated: 57.23 5.81 11.61 measured: 57.23 5.92 10.95FAB mass spectrum: M + 1 (871.5)

[0037] (Example of Detection)

[0038]FIG. 2 shows detailed results of the detection of a hairpinstructure composed of GCGAAAAACGC. A gold electrode modified with ahairpin type DNA (5′-HS-GCGAAAAACGC-3′ was dipped in solution containing10 mM of phosphate buffered saline (pH 7.0), 10 mM of KCl and 0.1 mM ofCNDIFc. Ag/AgCl standard electrode (reference electrode) and a counterelectrode of platinum were used to measure a cyclic voltamogram. Theresults were shown in graph (b) in FIG. 2. 1.2 μA of a response currentwas gained at 572 mV. That is, 20 pmol of hairpin structures provided aresponse current of 1.2 μA. Further, in this system, several femtomoleof hairpin structure type DNA's were detectable. A response current at−457 mV was shown responding to the presence of naphthalene diimide.

[0039] Further, the above experiment was carried out using a goldelectrode without any modification, providing results shown in graph(a). The above response current at 572 mV was not observed. Thedisappearance of the response current is reasonable, because ferrocenylgroups and naphthalene diimide moieties in CNDIFc's form charge transfercomplexes in solution. Moreover, the response current corresponding tothe presence of naphthalene diimide shifted to −312 mV and its amplitudewas below ½ of that of the response current shown in the graph (b). Suchresults indicates that a charge transfer complex, with a chargetransferred from ferrocene to naphthalene diimide, was formed inCNDIFc's in solution.

[0040] Contrary to this, in graph (b), current response at 572 mV wasshown. This response may be explained as follows. Due to the interactionbetween CNDIFc and the structural site, bases constituting nucleic acidsin the site are attached or sandwiched in CNDIFc's, inhibiting the aboveintermolecular charge transfer to induce measurable response current at572 mV corresponding to ferrocene.

[0041] Further, in the above experiment, the hairpin structure DNA washybridized with an oligonucleotide (5′-GCGTTTTTCGC-3′) complementary tothe hairpin DNA to cancel the hairpin structure site. The results wereshown in graph (c), in which current response at 572 mV was disappeared.The results show that, even when a double stranded nucleic acid sitecoexists with a specific single stranded nucleic acid site. the specificsingle stranded nucleic acid region may be detected. Further, theresults definitely confirms that CNDIFc specifically binds to a highlyordered structural site of a single stranded nucleic acid and therebyprovides current response on electrodes.

[0042] As described above, by contacting the inventive cyclic ligandcontaining naphthalene diimide moiety and ferrocenyl group with aworking electrode modified with a gene and measuring its electrochemicalbehavior or response currents by means of a cyclic voltamogram ordifferential pulse voltamogram, whether a highly ordered structural siteof a single stranded nucleic acid is present in the gene (a DNA or RNA)or not, and/or the content of the site may be evaluated. Particularly,the inventive detection probe may show high sensitivity, because itprovides an electrochemical response due to the inhibition of chargetransfer between ferrocenyl group and naphthalene diimide moiety, onlywhen the probe binds with the target site.

[0043] Although particular embodiments of the invention has beendescribed above, it is understood that the embodiments may be modifiedwithout departing from the scope of the invention, which is limited onlyby the appended claims.

1. A probe for detecting a highly ordered structural site of a nucleicacid of a gene by specifically binding with the structural site togenerate an electrochemical response, the probe comprising a cyclicligand containing ferrocenyl group and a DNA threading intercalatingmoiety.
 2. The probe as claimed in claim 1, wherein the cyclic ligandfurther comprises two linker moiety each having two terminal aminogroups, each linker moiety is connected with the DNA threadingintercalating moiety through one of its terminal amino groups, and eachlinker moiety is connected with the ferrocenyl group through the otherof its terminal amino groups.
 3. The probe as claimed in claim 2,wherein the linker moiety is a residual group of an amine having theterminal amino groups.
 4. The probe as claimed in claim 3, wherein theamine comprises another amino group, and two alkyl groups each bondedwith each terminal amino group and bonded with another amino group. 5.The probe as claimed in claim 4, wherein the another amino group ispiperazinyl group.
 6. The probe as claimed in claim 4, wherein the alkylgroup has 1 to 6 carbon atoms.
 7. The probe as claimed in claim 6,wherein the alkyl group is selected from a group consisting of ethylgroup and propyl group.
 8. The probe as claimed in claim 3, wherein theamine is selected from a group consisting of1,4-bis(3-aminopropyl)piperazine, 1,1′-bis(3-aminopropyl)methylamine,1,1′-bis(2-aminoethyl)amine, 1,1′-bis(3-aminopropyl)amine, spermine andspermidine.
 9. The probe as claimed in claim 2, wherein the DNAthreading intercalating moiety comprises an aromatic group selected froma group consisting of 1,4,5,8-tetrasubstituted naphthalene,9,10-disubstituted anthracene, and 1,5-disubstituted anthraquinone. 10.The probe as claimed in claim 9, wherein the aromatic group is1,4,5,8-tetrasubstituted naphthalene.
 11. The probe as claimed in claim9 or 10, wherein the DNA threading intercalating moiety furthercomprises carbonyl groups, iminomethylene groups, or thiocarbonylgroups, through which the DNA threading intercalating moiety is bondedwith the terminal amino groups.
 12. The probe as claimed in claim 1,wherein the structural site is a high-order structure in a DNA or RNA.13. The probe as claimed in claim 1, wherein the structural site isselected from a group consisting of a mismatch structure in an oncogenicDNA, a hairpin structure of a viral RNA and a bulge structure.
 14. Amethod for detecting a highly ordered structural site of a nucleic acidof a gene, the method comprising: contacting a gene with the probe asclaimed in claim 1 or 2 to generate an electrochemical response; anddetecting the electrochemical response.
 15. A device for detecting ahighly ordered structural site of a nucleic acid of a gene using theprobe as claimed in claim 1 or 2, the device comprising: a container, asolution for dissolving the prove, the solution being held in thecontainer, a working electrode modified with a gene, the workingelectrode dipped in the solution in the container, and a counterelectrode dipped in the solution in the container.